Seasonal population dynamics and the genetic structure of the mosquito vector Aedes aegypti in Sao Paulo, Brazil

Population genetic studies of insect vectors can generate knowledge to improve epidemiological studies focused on the decrease of pathogen transmission. In this study, we used nine SNPs across the Aedes aegypti genome to characterize seasonal population variations of this important dengue vector. Mosquito samples were obtained by ovitraps placed over Botucatu SP from 2005 to 2010. Our data show that, regardless of the large variation in mosquito abundance (deduced from the number of eggs obtained from ovitraps), the effective population size remained stable over the years. These results suggest that Ae. aegypti is able to maintain a sufficiently large active breeding population during the dry season to keep genetic frequencies stable. These results open new perspectives on mosquito survey and control methods.publishersversionpublishe

Primeiro isolamento de Dengue 4 no Estado de São Paulo, Brasil, 2011

We report the first isolation of Dengue virus 4 (DENV-4) in the state of São Paulo, from two patients - one living in São José do Rio Preto and the other one in Paulo de Faria, both cities located in the Northwest region of the state. The virus isolations were accomplished in the clone C6/36 Aedes albopictus cell line, followed by indirect immunofluorescence assays, performed with type-specific monoclonal antibodies that showed positive reactions for DENV-4. The results were confirmed by Nested RT-PCR and Real-Time RT-PCR assays. The introduction of DENV-4 in a country that already has to deal with the transmission of three other serotypes increases the possibility of the occurrence of more severe cases of the disease. The importance of early detection of dengue cases, before the virus spreads and major outbreaks occur, should be emphasized.Relatamos o primeiro isolamento do vírus Dengue 4 (DENV-4) no Estado de São Paulo, de dois pacientes residentes em São José do Rio Preto e Paulo de Faria, ambos municípios localizados na região Noroeste do Estado. O isolamento do vírus foi realizado em clone C6/36, linhagem de células de Aedes albopictus seguido por imunofluorescência indireta, realizada com anticorpos monoclonais tipo específicos, que apresentou reação positiva para DENV-4. Os resultados foram confirmados por testes de Nested RT-PCR e RT-PCR em Tempo Real. A introdução do DENV-4 no país, com uma população suscetível a esse vírus e que já convive com a transmissão de outros três sorotipos, aumenta a possibilidade da ocorrência de casos mais graves da doença. Deve ser enfatizada a importância da detecção precoce de casos de dengue, antes que ocorra a propagação do vírus e que surtos importantes aconteçam

Dengue Virus Type 4 Phylogenetics in Brazil 2011: Looking beyond the Veil

Dengue Fever and Dengue Hemorrhagic Fever are diseases affecting approximately 100 million people/year and are a major concern in developing countries. In the present study, the phylogenetic relationship of six strains of the first autochthonous cases of DENV-4 infection occurred in Sao Paulo State, Parana State and Rio Grande do Sul State, Brazil, 2011 were studied. Nucleotide sequences of the envelope gene were determined and compared with sequences representative of the genotypes I, II, III and Sylvatic for DEN4 retrieved from GenBank. We employed a Bayesian phylogenetic approach to reconstruct the phylogenetic relationships of Brazilian DENV-4 and we estimated evolutionary rates and dates of divergence for DENV-4 found in Brazil in 2011. All samples sequenced in this study were located in Genotype II. The studied strains are monophyletic and our data suggest that they have been evolving separately for at least 4 to 6 years. Our data suggest that the virus might have been present in the region for some time, without being noticed by Health Surveillance Services due to a low level of circulation and a higher prevalence of DENV-1 and DENV- 2

Desenvolvimento de metodologia high-throughput para estudo populacional do mosquito Aedes aegypti e comparação de dados de genes mitocondriais

O Aedes aegypti, culicídeo de hábitos diurnos, é originário do continente africano e está globalmente distribuído pelos trópicos em associação com as populações humanas. É considerado de grande importância epidemiológica por ser o principal vetor dos quatro sorotipos do vírus da dengue e da febre amarela. Uma das primeiras detecções da presença do mosquito no Estado de São Paulo aconteceu na década de 80 na cidade de Santos. Atualmente não há disponível nenhuma vacina ou medicamento eficiente contra a dengue, assim o controle da doença está restrito ao controle do vetor. Uma das alternativas de controle e entendimento das relações vetor-patógeno-homem se baseiam no desenvolvimento de ferramentas moleculares que utilizam técnicas baseadas em PCR, as quais têm possibilitado o estudo genético das populações do Ae. aegypti. Em tais estudos, vários marcadores foram envolvidos, tais como os SNPs (Single Nucleotide Polymorphisms) e marcadores mitocondriais. Nós desenvolvemos ensaios utilizando metodologia TaqMan® para o estudo genético populacional de duas populações do mosquito Aedes aegypti de cidades portuárias do Estado de São Paulo, utilizando nove marcadores SNPs. Verificamos que esta metodologia é reprodutível, rápida, de baixo custo e eficiente para estudos em larga escala. Pela análise AMOVA encontramos uma baixa, mas significativa diferenciação genética entre as populações do estudo (FST = 0,0324; P < 0,01), e uma alta taxa de migrantes por geração (8,72 entre as populações 2007 e 5,39 entre as populações 2008), indicando fluxo gênico entre as populações. A análise implementada no software Structure 2.3.1, evidenciou a existência de três clusters baseados em semelhanças genotípicas, distribuídos em dois grupos, confirmando uma moderada estruturação populacional. Verificamos através da análise de fragmentos...Aedes aegypti, is a diurnal mosquito, originated from the African continent and is globally distributed through the tropics in association with human populations. It is considered of great epidemiological importance for being the main vector of the four serotypes of Dengue and Yellow Fever. One of the first detections of the presence of the mosquito in the State of São Paulo happened in the 80's, in the city of Santos. Currently there is no available vaccine or effective medicine against dengue fever, and disease control is restricted to vector control. An alternative to control and understanding of vectorpathogen- man relationships are based on the development of molecular tools that use PCR-based techniques, which have enabled the genetic study of populations of Ae. aegypti. In such studies, several markers were involved, such as SNPs (Single Nucleotide Polymorphisms) and mitochondrial ones. We have developed assays using TaqMan® methodology for population genetic studies of two populations of Aedes aegypti from the port cities of São Paulo, using nine SNPs markers. We found that this methodology is reproducible, fast, inexpensive and efficient for large-scale studies. AMOVA analysis found a low but significant genetic differentiation between the studied populations (FST = 0.0324, P <0.01), and a high rate of migrants per generation (8.72 among populations in 2007 and 5.39 among populations in 2008), indicating gene flow between populations. The analysis implemented in software Structure 2.3.1, revealed the existence of three clusters based on genotypic similarities, divided into two groups, confirming a moderate population structure. We verified through the analysis of the mitochondrial gene fragments NADH dehydrogenase subunit 4 (ND4) a high genetic differentiation between the two study populations (FST = 0.18034, P <0.01), and a rate of migrants per generation considered high... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Caracterização das respostas transcricionais e microbiomas de populações naturais do mosquito Aedes aegypti com diferentes níveis de suscetibilidade ao vírus dengue

Dengue is the arbovirosis of greater growth in recent years, reflecting on social and economic impacts due to the high morbidity and mortality rates triggered by infection. Dengue has Aedes aegypti as its primary vector, which is distributed throughout tropical and subtropical regions of the planet. Due to its anthropophilic and haematophagic habit, rapid development and specific behavioral characteristics, it is an excellent dengue virus transmitter. Control measurements are restricted to eliminating the mosquito vector since neither specific treatments nor tetravalent vaccines are yet commercially available. One feature that determines the spread of the disease is the high vector competence of its vector mosquitoes, which has been associated to its genetic factors and to its gut microbiota. The present study evaluated genetic and microbial factors related to vector competence of natural populations of Ae. Aegypti with different levels of susceptibilities to dengue virus serotype 4 (DENV-4). For that, we evaluated the susceptibility to DENV-4 infection of two field collected mosquito populations (Botucatu-SP and Neópolis-SE) through infection assays and relative quantification by qPCR. Differential gene expression and gut microbial diversity analyses were performed using next-generation sequencing (RNA-seq and 16S rRNA gene sequencing) through Illumina HiScanSQ and MiSeq platforms, respectively. The populations presented different susceptibility to DENV-4, of which Botucatu showed higher resistance to infection when compared to Neópolis. Differential gene expression analysis revealed that there was little gene modulation in response to DENV infection in Botucatu, while Neópolis showed consistent modulation on several genes related to immune pathways or digestive processes. When comparing to other studies we found that the Toll immune pathway is being activated in this population after infection. However, the comparison of ...Dengue é a arbovirose de maior crescimento nos últimos anos, repercutindo em impactos sociais e econômicos devido às altas taxas de morbidade e mortalidade desencadeadas pela infecção. O dengue tem como principal vetor o mosquito Aedes aegypti, distribuído em toda a faixa tropical e subtropical do planeta. Por apresentar hábito hematofágico antropofílico, rápido desenvolvimento e características comportamentais específicas, é um excelente transmissor do vírus dengue. Medidas de controle estão restritas à eliminação do mosquito vetor, uma vez que um tratamento específico ou uma vacina que previna simultaneamente a infecção pelos quatro sorotipos deste arbovírus ainda não estão disponíveis à população. Uma característica que determina a disseminação de doenças é a alta competência vetorial de seus mosquitos transmissores, que tem sido associada à fatores genéticos do mosquito bem como à microbiota intestinal do inseto. O presente estudo avaliou fatores genéticos e microbianos relacionados à competência vetorial de populações naturais de Ae. aegypti com diferenças na susceptibilidades ao vírus dengue sorotipo 4 (DENV-4). Para isso, foi avaliada a susceptibilidade à infecção pelo DENV de duas populações naturais deste inseto (Botucatu-SP e Neópolis-SE) através de ensaios de infecção e quantificação relativa por PCR em tempo real. A expressão gênica diferencial, bem como a diversidade microbiana intestinal, foi realizada através do sequenciamento de nova geração (RNA-seq e sequenciamento do gene 16S rRNA) através das plataformas Illumina HiScan™SQ e MiSeq, respectivamente. Verificamos que as populações estudadas apresentam diferenças na susceptibilidades ao DENV-4 onde Botucatu apresentou maior resistência à infecção quando comparada à Neópolis. Pela análise de expressão gênica diferencial verificamos que houve pouca modulação genica na população de Botucatu em resposta a...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Seasonal population dynamics and the genetic structure of the mosquito vector Aedes aegypti in São Paulo, Brazil

Population genetic studies of insect vectors can generate knowledge to improve epidemiological studies focused on the decrease of pathogen transmission. In this study, we used nine SNPs across the Aedes aegypti genome to characterize seasonal population variations of this important dengue vector. Mosquito samples were obtained by ovitraps placed over Botucatu SP from 2005 to 2010. Our data show that, regardless of the large variation in mosquito abundance (deduced from the number of eggs obtained from ovitraps), the effective population size remained stable over the years. These results suggest that Ae. aegypti is able to maintain a sufficiently large active breeding population during the dry season to keep genetic frequencies stable. These results open new perspectives on mosquito survey and control methods.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters

Hepatozoon spp. are commonly found infecting snakes. Since the latter are parasitized by diverse forms and data in the literature show divergence, we studied Hepatozoon spp. diversity on Crotalus durissus terrificus snakes using both molecular and morphological approaches. Naturally infected animals were employed. Blood was collected, blood smears were prepared and an aliquot was stored at -20. °C for DNA extraction. Five specimens of C. durissus terrificus were selected, each of them infected with one gamont type. Morphological and morphometric analyses of the found gamonts led to their grouping into three populations. For molecular characterization, seven oligonucleotide pairs that amplify distinct regions of rDNA gene were tested by adopting the PCR technique. Only the oligonucleotide pairs HepF300/Hep900 and HEMO1/HEMO2 were efficient in amplifying and distinguishing different isolates of Hepatozoon spp. from snakes. The better results were obtained when both oligonucleotide pairs were used in association. Based on the molecular and morphologic differences, three new species were proposed: Hepatozoon cuestensis sp. nov.; Hepatozoon cevapii sp. nov. and Hepatozoon massardii sp. nov. This is the first description of new Hepatozoon species from snakes, based on molecular characterization and morphological data, in South America. © 2013 Elsevier Inc